Generation and correction of a humanized mouse model with a deletion of dystrophin exon 44

ABSTRACT

Duchenne muscular dystrophy (DMD), which affects 1 in 5,000 male births, is one of the most common genetic disorders of children. This disease is caused by an absence or deficiency of dystrophin protein in striated muscle. The major DMD deletion “hot spots” are found between exon 6 to 8, and exons 45 to 53. Here, a “humanized” mouse model is provided that can be used to test a variety of DMD exon skipping strategies. Among these are, CRISPR/Cas9 oligonucleotides, small molecules or other therapeutic modalities that promote exon skipping or micro dystrophin mini genes or cell based therapies. Methods for restoring the reading frame of exon 44 deletion via CRISPR-mediated exon skipping in the humanized mouse model, in patient-derived iPS cells and ultimately, in patients using various delivery systems are also contemplated. The impact of CRISPR technology on DMD is that gene editing can permanently correct mutations.

PRIORITY CLAIM

The present application is a divisional of U.S. application Ser. No.15/914,728, filed Mar. 7, 2018, which claims benefit of priority to U.S.Provisional Application Ser. No. 62/468,154, filed Mar. 7, 2017, theentire contents of each of which are hereby incorporated by reference intheir entirety.

FEDERAL FUNDING SUPPORT CLAUSE

This invention was made with government support under grant no. U54 HD087351 awarded by National Institutes of Health. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 12, 2020, isnamed UTSDP3136USD1.txt and is 17 kilobytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates to the fields of molecular biology,medicine and genetics. More particularly, the disclosure relates to theuse of genome editing to create humanized animal models for differentforms of Duchenne muscular dystrophy (DMD), each containing distinct DMDmutations.

BACKGROUND

Muscular dystrophies (MD) are a group of more than 30 genetic diseasescharacterized by progressive weakness and degeneration of the skeletalmuscles that control movement. Duchenne muscular dystrophy (DMD) is oneof the most severe forms of MD that affects approximately 1 in 5000 boysand is characterized by progressive muscle weakness and premature death.Cardiomyopathy and heart failure are common, incurable and lethalfeatures of DMD. The disease is caused by mutations in the gene encodingdystrophin (DMD), a large intracellular protein that links thedystroglycan complex at the cell surface with the underlyingcytoskeleton, thereby maintaining integrity of the muscle cell membraneduring contraction. Mutations in the dystrophin gene result in loss ofexpression of dystrophin causing muscle membrane fragility andprogressive muscle wasting.

Despite intense efforts to find cures through a variety of approaches,including myoblast transfer, viral delivery, andoligonucleotide-mediated exon skipping, there remains no cure for anytype of muscular dystrophy. The present inventors recently usedclustered regularly interspaced short palindromic repeat/Cas9(CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene(DMD) mutation in postnatal mdx mice, a model for DMD. In vivoAAV-mediated delivery of gene-editing components successfully removedthe mutant genomic sequence by exon skipping in the cardiac and skeletalmuscle cells of mdx mice. Using different modes of AAV9 delivery, theinventors restored dystrophin protein expression in cardiac and skeletalmuscle of mdx mice. The mdx mouse model and the correction exon 23 usingAAV delivery of myoediting machinery has been useful to show proof-ofconcept of exon skipping approach using several cuts in genomic regionencompassing the mutation in vivo. Recent work with ΔEx50 mouse modeldemonstrated an optimized the method for dystrophin reading framecorrection using a single guide RNA that created reframing mutations andallowed permanent skipping of exon 51. However, there is a lack of othermodels for the various known DMD mutations, and for new mutations thatcontinue to be discovered.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amouse whose genome comprises a deletion of exon 44 of the dystrophingene resulting in an out of frame shift and a premature stop codon inexon 45. These mice are highly useful because they contain the secondmost prominent dystrophin mutation in human, representing ˜12% ofpatients with DMD. The genome of the mouse may further comprise areporter gene located downstream of and in frame with exon 79 of thedystrophin gene, and upstream of a dystrophin 3′-UTR, wherein saidreporter gene is expressed when exon 79 is translated in frame with exon46. The reporter gene may be luciferase. The genome of the mouse mayfurther comprise a protease coding sequence upstream of and in framewith said reporter gene, and downstream of and in frame with exon 79.The protease may be autocatalytic, such as 2A protease. The mouse may beheterozygous for said deletion, or homozygous for said deletion. Themouse may exhibit increased creatine kinase levels, and/or may notexhibit detectable dystrophin protein in heart or skeletal muscle.

Also provided is a method of producing the mouse described abovecomprising (a) contacting a fertilized oocyte with CRISPR/Cas9 elementsand two single guide RNA (sgRNA) targeting sequences flanking exon 44,thereby creating a modified oocyte, wherein deletion of exon 44 byCRISPR/Cas9 results in an out of frame shift and a premature stop codonin exon 45; (b) transferring said modified oocyte into a recipientfemale. The oocyte genome may comprise a dystrophin gene having areporter gene located downstream of and in frame with exon 79 of saiddystrophin gene, and upstream of a dystrophin 3′-UTR, wherein saidreporter gene is expressed when exon 79 is translated in frame with exon43. The reporter gene may be luciferase. The oocyte genome may furthercomprise a protease coding sequence upstream of and in frame with saidreporter gene, and downstream of and in frame with exon 79. The proteasemay be autocatalytic, such as 2A protease. The mouse may be heterozygousfor said deletion, or homozygous for said deletion. The mouse mayexhibit increased creatine kinase levels and/or may not exhibitdetectable dystrophin protein in heart or skeletal muscle.

In another embodiment, there is provided an isolated cell obtained fromthe mouse described above. The genome of the cell may further comprise areporter gene located downstream of and in frame with exon 79 of thedystrophin gene, and upstream of a dystrophin 3′-UTR, wherein saidreporter gene is expressed when exon 79 is translated in frame with exon43. The reporter gene may be luciferase. The genome of the cell mayfurther comprise a protease coding sequence upstream of and in framewith said reporter gene, and downstream of and in frame with exon 79.The protease may be autocatalytic, such as 2A protease. The cell may beheterozygous for said deletion, or homozygous for said deletion.

In a further embodiment, there is provided a mouse produced by a methodcomprising the steps of (a) contacting a fertilized oocyte withCRISPR/Cas9 elements and two single guide RNA (sgRNA) targetingsequences flanking exon 44, thereby creating a modified oocyte, whereindeletion of exon 44 by CRISPR/Cas9 results in an out of frame shift anda premature stop codon in exon 45; (b) transferring said modified oocyteinto a recipient female.

These mice provide an important system for assessing the efficacy of avariety of therapeutic analogues for correction of DMD mutation. In oneembodiment, CRISPR/Cas9 can be used to skip exon 45, putting thedystrophin protein back in frame. The mice allow for rapid optimizationof the method. In other embodiments, the mice can be used to testexon-skipping oligonucleotides or small molecules or other therapeuticmodalities in a “humanized” system. In still a further embodiment, thereis provided a method of screening a candidate substance for DMDexon-skipping activity comprising (a) contacting a mouse according toclaim 1 with a candidate substance; and (b) assessing in frametranscription and/or translation of exon 79, wherein the presence of inframe transcription and/or translation of exon 79 indicates saidcandidate substance exhibits exon-skipping activity.

A further embodiment comprises an isolated nucleic acid comprising asequence as set forth below:

SEQ ID ID Sequence (5′-3′) NO. Ex45-gRNA#3-Top CACCGCGCTGCCCAATGCCATCCTG1 Ex45-gRNA#3-Bot AAACCAGGATGGCATTGGGCAGCGC 2 Ex45-gRNA#4-TopCACCGCTTACAGGAACTCCAGGA 3 Ex45-gRNA#4-Bot AAACTCCTGGAGTTCCTGTAAGC 4Ex45-gRNA#5-Top CACCGAGGAACTCCAGGATGGCATT 5 Ex45-gRNA#5-BotAAACAATGCCATCCTGGAGTTCCTC 6 Ex45-gRNA#6-Top CACCGCGCTGCCCAATGCCATCC 7Ex45-gRNA#6-Bot AAACGGATGGCATTGGGCAGCGC 8 Ex45-gRNA#4-mDmd-CACCGGCTTACAGGAACTCCAGGA 27 20-Top Ex45-gRNA#4-mDmd-AAACTCCTGGAGTTCCTGTAAGCC 28 20-Bot Ex45-gRNA#4-DMD-CACCGATCTTACAGGAACTCCAGGA 29 20-Top Ex45-gRNA#4-DMD-AAACTCCTGGAGTTCCTGTAAGATC 30 20-Bot Ex43-gRNA#1-DMD-TopCACCGTTTTAAAATTTTTATATTA 31 Ex43-gRNA#1-DMD-Bot AAACTAATATAAAAATTTTAAAAC32 Ex43-gRNA#2-DMD-Top CACCGTTTTATATTACAGAATATAA 33 Ex43-gRNA#2-DMD-BotAAACTTATATTCTGTAATATAAAAC 34 Ex43-gRNA#4-DMD-TopCACCGTATGTGTTACCTACCCTTGT 35 Ex43-gRNA#4-DMD-BotAAACACAAGGGTAGGTAACACATAC 36 Ex43-gRNA#6-DMD-TopCACCGTACAAGGACCGACAAGGGT 37 Ex43-gRNA#6-DMD-Bot AAACACCCTTGTCGGTCCTTGTAC38Also provided is a double-stranded nucleic acid formed by hybridizationof SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ IDNO: 7 and 8, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, SEQ ID NO: 31and 32, SEQ ID NO: 33 and 34, SEQ ID NO: 35 and 36, or SEQ ID NO: 37 and38, and an expression construct comprising a nucleic acid formed byhybridization of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30,SEQ ID NO: 31 and 32, SEQ ID NO: 33 and 34, SEQ ID NO: 35 and 36, SEQ IDNO: 37 and 38, such as a viral or non-viral vector. Additionally, a kitcomprising one or more isolated nucleic acids as set forth below isdescribed:

SEQ ID ID Sequence (5′-3′) NO. Ex45-gRNA#3-Top CACCGCGCTGCCCAATGCCATCCTG1 Ex45-gRNA#3-Bot AAACCAGGATGGCATTGGGCAGCGC 2 Ex45-gRNA#4-TopCACCGCTTACAGGAACTCCAGGA 3 Ex45-gRNA#4-Bot AAACTCCTGGAGTTCCTGTAAGC 4Ex45-gRNA#5-Top CACCGAGGAACTCCAGGATGGCATT 5 Ex45-gRNA#5-BotAAACAATGCCATCCTGGAGTTCCTC 6 Ex45-gRNA#6-Top CACCGCGCTGCCCAATGCCATCC 7Ex45-gRNA#6-Bot AAACGGATGGCATTGGGCAGCGC 8 Ex45-gRNA#4-mDmd-CACCGGCTTACAGGAACTCCAGGA 27 20-Top Ex45-gRNA#4-mDmd-AAACTCCTGGAGTTCCTGTAAGCC 28 20-Bot Ex45-gRNA#4-DMD-CACCGATCTTACAGGAACTCCAGGA 29 20-Top Ex45-gRNA#4-DMD-AAACTCCTGGAGTTCCTGTAAGATC 30 20-Bot Ex43-gRNA#1-DMD-CACCGTTTTAAAATTTTTATATTA 31 Top Ex43-gRNA#1-DMD-AAACTAATATAAAAATTTTAAAAC 32 Bot Ex43-gRNA#2-DMD-CACCGTTTTATATTACAGAATATAA 33 Top Ex43-gRNA#2-DMD-AAACTTATATTCTGTAATATAAAAC 34 Bot Ex43-gRNA#4-DMD-CACCGTATGTGTTACCTACCCTTGT 35 Top Ex43-gRNA#4-DMD-AAACACAAGGGTAGGTAACACATAC 36 Bot Ex43-gRNA#6-DMD-CACCGTACAAGGACCGACAAGGGT 37 Top Ex43-gRNA#6-DMD-AAACACCCTTGTCGGTCCTTGTAC 38 Botor an expression vector coding therefor.

Still a further embodiment comprises a method of correcting a dystrophingene defect in Exon 45 of the DMD gene in a subject comprisingcontacting a cell in said subject with Cpf1 or Cas9 and a DMD guide RNAas defined above, resulting in selective skipping of a mutant DMD exon.The cell may be a muscle cell, a satellite cell, or an iPSC/iCM. Cpf1and/or DMD guide RNA may be provided to said cell through expressionfrom one or more expression vectors coding therefor, such as a viralvector (e.g., adeno-associated viral vector) or as a non-viral vector.Cpf1 or Cas9 may be provided to said cell as naked plasmid DNA orchemically-modified mRNA.

The method may further comprise contacting said cell with asingle-stranded DMD oligonucleotide to effect homology directed repairor non-homologous end joining (NHEJ). Cpf1 or Cas9, DMD guide RNA and/orsingle-stranded DMD oligonucleotide, or expression vectors codingtherefor, may be provided to said cell in one or more nanoparticles.Cpf1 or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotidemay be delivered directly to a muscle tissue, such as tibialis anterior,quadricep, soleus, diaphragm or heart. Cpf1 or Cas9, DMD guide RNAand/or single-stranded DMD oligonucleotide may be deliveredsystemically.

The subject may exhibit normal dystrophin-positive myofibers and/ormosaic dystrophin-positive myofibers containing centralized nuclei. Thesubject may exhibit a decreased serum CK level as compared to a serum CKlevel prior to contacting. The subject may exhibit improved gripstrength as compared to a serum CK level prior to contacting. Thecorrection may be permanent skipping of said mutant DMD exon, or morethan one mutant DMD exon. The Cpf1 or Cas9 and/or DMD guide RNA may bedelivered to a human iPS cell with an adeno-associated viral vector.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-H. Exon 44 deleted DMD patient iPSC-derived cardiomyocytesexpress dystrophin after CRISPR/Cas9 mediated genome editing by exonskipping. (FIG. 1A) A DMD deletion of exon 44 results in splicing ofexon 43 to 45, generating an out-of-frame mutation of dystrophin.Disruption of the splice acceptor of exon 45 results in splicing of exon43 to 46 and restoration of the protein reading frame. (FIG. 1B)Illustration of sgRNAs targeting exon 45 splice acceptor site. The PAM(red) of the sgRNAs is located near the exon45 splice acceptor site. The100% human and mouse conserved sequence is shaded in light yellow. Exonsequence is bold upper case. Intron sequence is lower case. (SEQ ID NOS:22-23) (FIG. 1C) T7E1 assay using 10T1/2 mouse cells and 293 human cellstransfected with spCas9 and exon45 sgRNA3 (G3), sgRNA4 (G4), sgRNA5 (G5)or sgRNA6 (G6) shows cleavage of the DMD locus at intron-exon junctionof exon45. Red arrowheads denote cleavage products. (FIG. 1D) T7E1 assayusing iPS cells transfected with spCas9 and exon 45 sgRNA3 (G3) showscleavage of the DMD locus at intron-exon junction of exon45. Redarrowheads denote cleavage products. (FIG. 1E) PCR products of genomicDNA isolated from single clones of exon 44 deleted DMD-iPSCs transfectedwith a plasmid expressing spCas9 and exon 45 sgRNA3 (G3). Sequence ofthe PCR products of each clone shows deletions from the 3′-end of intron44 to the 5′-end of exon 45. This confirms removal of the “ag” spliceacceptor of exon 45. The sequence of the uncorrected allele is shownabove that of the exon 45-skipped allele. (SEQ ID NOS: 24-26) (FIG. 1F)Western blot analysis shows restoration of dystrophin expression in exon45-skipped single clones. Vinculin was used as a loading control. (FIG.1G) Illustration of an AAV9 plasmid encoding three spCas9 sgRNAs drivenby the U6, H1, and 7SK promoters. The plasmid also encodes a GFP drivenby CK8 promoter. A separate AAV9 plasmid encodes spCas9 driven by CK8promoter. (FIG. 1H) T7E1 assay using C2C12 mouse cells transfected withspCas9 and exon 45 sgRNA3 (G3), exon 45 sgRNA4 (G4), AAV9-CK8-exon 45sgRNA3 or sgRNA4 shows cleavage of the DMD locus at intron-exon junctionof exon45. Red arrowheads denote cleavage products.

FIGS. 2A-G. “Humanized”-ΔEx44 mouse model. (FIG. 2A) Outline of theCRISPR/Cas9 strategy used for generation of the mice. (FIG. 2B) Outlineof the CRISPR/Cas9 strategy to deplete exon 44. T7E1 assay using 10T1/2mouse cells transfected with spCas9 with different sgRNAs targeting 5′end (In44-1, In44-2 or In44-3) and 3′ end (In44-4, In44-5, In44-6) ofexon 44 shows different cleavage efficiency at the Dmd exon 44. Redarrowheads show cleavage products of genome editing. (FIG. 2C) PCRgenotyping of 10 F1 pups shows efficient exon 44 depletion byCRISPR/Cas9-mediated genome editing. The lower band (red arrowheads)shows exon44 deletion. (FIG. 2D) Serum creatine kinase (CK), a marker ofmuscle dystrophy that reflects muscle damage and membrane leakage wasmeasured in wild type (WT), ΔEx44, BL/10 and mdx mice. (FIG. 2E) Westernblot analysis shows loss of dystrophin expression in heart, TA muscle,and gastrocnemius/plantaris (G/P) muscle of ΔEx44 mice. Vinculin wasused as a loading control. (FIG. 2F) Dystrophin staining of TA,diaphragm and cardiac muscle. (FIG. 2G) Hematoxylin and eosin (H&E)staining of TA, diaphragm and cardiac muscle.

FIGS. 3A-F. Identification of optimal sgRNAs for CRISPR/Cas9 correctionof DMD Exon 44 deletions. (FIG. 3A) A DMD deletion of exon 44 results insplicing of exon 43 to 45, generating an out-of-frame mutation ofdystrophin. Disruption of the splice junction of exon 43 or exon 45results in splicing of exon 42 to 45 or exon 43 to 46 and restoration ofthe protein reading frame. Alternatively, gene editing results inrestoration of the protein reading frame. (FIG. 3B) Illustration of the100% human and mouse conserved sequence at the intron-exon junction ofexon 45. The conserved region is shaded in light blue. FIG. 3B disclosesSEQ ID NOS 55-56, respectively, in order of appearance. (FIG. 3C)Illustration of sgRNAs targeting exon 45 splice acceptor site. The PAM(red) of the sgRNAs is located near the exon45 splice acceptor site. The100% human and mouse conserved sequence is shaded in light blue. Exonsequence is bold upper case. Intron sequence is lower case. FIG. 3Cdiscloses SEQ ID NO: 57. (FIG. 3D) Illustration of sgRNAs targeting exon43 splice acceptor and donor site. The PAM (red) of the sgRNAs islocated near the exon43 splicing junctions. Exon sequence is bold uppercase. Intron sequence is lower case. FIG. 3D discloses SEQ ID NOS 58-59,respectively, in order of appearance. (FIG. 3E) T7E1 assay using 293human cells transfected with spCas9 and exon43 sgRNA1 (G1), sgRNA2 (G2),sgRNA4 (G4) or sgRNA6 (G6) shows cleavage of the DMD locus atintron-exon junctions of exon43. Red arrowheads denote cleavageproducts. (FIG. 3F) T7E1 assay using 10T1/2 mouse cells and 293 humancells transfected with spCas9 and exon45 sgRNA3 (G3), sgRNA4 (G4),sgRNA5 (G5) or sgRNA6 (G6) shows cleavage of the DMD locus atintron-exon junction of exon45. Red arrowheads denote cleavage products.

FIGS. 4A-D. Exon 44 deleted DMD patient iPSC-derived cardiomyocytesexpress dystrophin after CRISPR/Cas9 mediated genome editing by exonskipping (FIG. 4A) Schematic of the procedure for derivation and editingof patient-derived iPSCs. (FIG. 4B) Western blot analysis shows absenceof dystrophin in cardiomyocytes differentiated from two patient-derivediPSCs. (FIG. 4C) Western blot analysis shows restoration of dystrophinexpression in exon 45-edited and exon 43-edited cells. Vinculin isloading control. (FIG. 4D) Immunostaining shows restoration ofdystrophin expression in exon 45-edited and exon 43-edited cells.Dystrophin stains in red. Cardiac troponinl stains in green. Nucleusmarks by DAPI stains in blue.

FIGS. 5A-E. Characterization of ΔEx44 mice. (FIG. 5A) Outline of theCRISPR/Cas9 strategy used for generation of the mice. (FIG. 5B) Activityof serum creatine kinase (CK), a marker of muscle dystrophy thatreflects muscle damage and membrane leakage was measured in wild type(WT) and 4Ex44 mice. Maximal tetanic force (FIG. 5C), specific force(FIG. 5D), and forelimb grip strength (FIG. 5E) were reduced in 4Ex44mice compared to wild type (WT) mice, indicating decreased musclefunction.

FIGS. 6A-G. Correction of DMD exon 44 deletion in mice by intramuscularAAV9 delivery of gene editing components. (FIG. 6A) Immunostaining showsrestoration of dystrophin in TA muscle of Δ44 mice 3 weeks afterintramuscular injection of gene editing component carried by AAV9.Dystrophin stains in red. Nucleus marks by DAPI stains in blue. (FIG.6B) Hematoxylin and eosin (H&E) staining of TA and cardiac muscles inwildtype (WT), Δ44, and corrected Δ44 mice. (FIG. 6C) Western blotanalysis shows restoration of dystrophin expression in TA muscle andheart of Δ44 mice. Vinculin is loading control. (FIG. 6D) T7E1 assayshows cleavage of the DMD locus at intron-exon junction of exon45 in TAmuscle of corrected Δ44 mice. Red arrowheads show cleavage products ofgenome editing. (FIG. 6E) RT-PCR analysis of the TA muscles from WT, Δ44and Δ44 mice 3 weeks after intramuscular injection of gene editingcomponent carried by AAV9. Lower dystrophin bands indicate skipping ofexon 45. (FIG. 6F) Percentage of events detected at exon 45 afterAAV9-Cas9/exon45-sgRNA4 treatment using RT-PCR sequence analysis ofTOPO-TA (topoisomerase-based thymidine to adenosine) generated clones.RT-PCR products are divided into four groups: Not edited (NE), exon45-skipped (SK), reframed (RF), and out-of-frame (OF). (FIG. 6G)Sequence of the RT-PCR products of the WT, Δ44 and corrected Δ44 mice.Both exon 45-skipped and +1 reframed sequences are shown. FIG. 6Gdiscloses SEQ ID NOS 60-67, respectively, in order of appearance.

FIGS. 7A-C. Systemic AAV9 delivery of gene editing components to Δ44mice rescues dystrophin expression. Different AAV9-Cas9 andAAV9-exon45-sgRNA4 ratios were injected into Δ44 mice: 1:1.7 (5×10¹³vg/kg of AAV9-Cas9 to 8.5×10¹³ vg/kg of AAV9-exon45-sgRNA4); 1:2 (5×10¹³vg/kg of AAV9-Cas9 to 1×10¹⁴ vg/kg of AAV9-exon45-sgRNA4); 1:2.5 5×10¹³vg/kg of AAV9-Cas9 to 1.25×10¹³ vg/kg of AAV9-exon45-sgRNA4) and 1:5(5×10¹³ vg/kg of AAV9-Cas9 to 2×10¹⁴ vg/kg of AAV9-exon45-sgRNA4). (FIG.7A) Western blot analysis shows restoration of dystrophin expression inTA, diaphragm, triceps and cardiac muscles of Δ44 mice 4 weeks aftersystemic delivery of AAV9-Cas9 or AAV9-Cas9/exon45-sgRNA4. Vinculin wasused as a loading control. (FIG. 7B) Immunostaining shows restoration ofdystrophin in TA, diaphragm, triceps and cardiac muscles of Δ44 mice 4weeks after systemic delivery of AAV9-Cas9 or AAV9-Cas9/exon45-sgRNA4.Dystrophin stains in red. Nucleus marks by DAPI stains in blue. (FIG.7C) Reduction of serum creatine kinase activity in Δ44 mice 4 weeksafter systemic delivery of AAV9-Cas9 or AAV9-Cas9/exon45-sgRNA4.

DETAILED DESCRIPTION

DMD is a new mutation syndrome with more than 4,000 independentmutations that have been identified in humans (world-wide web atdmd.nl). The majority of patient's mutations carry deletions thatcluster in a hotspot, and thus a therapeutic approach for skippingcertain exon applies to large group of patients. The rationale of theexon skipping approach is based on the genetic difference between DMDand Becker muscular dystrophy (BMD) patients. In DMD patients, thereading frame of dystrophin mRNA is disrupted resulting in prematurelytruncated, non-functional dystrophin proteins. BMD patients havemutations in the DMD gene that maintain the reading frame allowing theproduction of internally deleted, but partially functional dystrophinsleading to much milder disease symptoms compared to DMD patients.

One the most common hot spots in DMD is the genetic region between exons44 and 51, where skipping of exon 45 would apply to ˜12% of the DMDpopulation. The instant disclosure demonstrates the efficiency ofCRISPR/Cas9 mediated correction of DMD mutations in patient-derived iPScells. To further assess the efficiency and optimizeCRISPR/Cas9-mediated exon skipping in vivo, a mimic of the human “hotspot” region was generated in a mouse model by deleting the exon 44using CRISPR/Cas9 system directed by two single guide RNAs (sgRNAs). TheΔEx44 mouse model exhibits dystrophic myofibers and increased serumcreatine kinase level, thus providing a representative model of DMD.These and other aspects of the disclosure are reproduced below.

I. DUCHENNE MUSCULAR DYSTROPHY

A. Background

Duchenne muscular dystrophy (DMD) is a recessive X-linked form ofmuscular dystrophy, affecting around 1 in 5000 boys, which results inmuscle degeneration and premature death. The disorder is caused by amutation in the gene dystrophin, located on the human X chromosome,which codes for the protein dystrophin. Dystrophin is an importantcomponent within muscle tissue that provides structural stability to thedystroglycan complex (DGC) of the cell membrane. While both sexes cancarry the mutation, females are rarely affected with the skeletal muscleform of the disease.

Mutations vary in nature and frequency. Large genetic deletions arefound in about 60-70% of cases, large duplications are found in about10% of cases, and point mutants or other small changes account for about15-30% of cases. Bladen et al. (2015), who examined some 7000 mutations,catalogued a total of 5,682 large mutations (80% of total mutations), ofwhich 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) wereduplications (1 exon or larger). There were 1,445 small mutations(smaller than 1 exon, 20% of all mutations), of which 358 (25%) weresmall deletions and 132 (9%) small insertions, while 199 (14%) affectedthe splice sites. Point mutations totaled 756 (52% of small mutations)with 726 (50%) nonsense mutations and 30 (2%) missense mutations.Finally, 22 (0.3%) mid-intronic mutations were observed. In addition,mutations were identified within the database that would potentiallybenefit from novel genetic therapies for DMD including stop codonread-through therapies (10% of total mutations) and exon skippingtherapy (80% of deletions and 55% of total mutations).

B. Symptoms

Symptoms usually appear in boys between the ages of 2 and 3 and may bevisible in early infancy. Even though symptoms do not appear until earlyinfancy, laboratory testing can identify children who carry the activemutation at birth. Progressive proximal muscle weakness of the legs andpelvis associated with loss of muscle mass is observed first. Eventuallythis weakness spreads to the arms, neck, and other areas. Early signsmay include pseudohypertrophy (enlargement of calf and deltoid muscles),low endurance, and difficulties in standing unaided or inability toascend staircases. As the condition progresses, muscle tissueexperiences wasting and is eventually replaced by fat and fibrotictissue (fibrosis). By age 10, braces may be required to aid in walkingbut most patients are wheelchair dependent by age 12. Later symptoms mayinclude abnormal bone development that lead to skeletal deformities,including curvature of the spine. Due to progressive deterioration ofmuscle, loss of movement occurs, eventually leading to paralysis.Intellectual impairment may or may not be present but if present, doesnot progressively worsen as the child ages. The average life expectancyfor males afflicted with DMD is around 25.

The main symptom of Duchenne muscular dystrophy, a progressiveneuromuscular disorder, is muscle weakness associated with musclewasting with the voluntary muscles being first affected, especiallythose of the hips, pelvic area, thighs, shoulders, and calves. Muscleweakness also occurs later, in the arms, neck, and other areas. Calvesare often enlarged. Symptoms usually appear before age 6 and may appearin early infancy. Other physical symptoms are:

-   -   Awkward manner of walking, stepping, or running—(patients tend        to walk on their forefeet, because of an increased calf muscle        tone. Also, toe walking is a compensatory adaptation to knee        extensor weakness.)    -   Frequent falls    -   Fatigue    -   Difficulty with motor skills (running, hopping, jumping)    -   Lumbar hyperlordosis, possibly leading to shortening of the        hip-flexor muscles. This has an effect on overall posture and a        manner of walking, stepping, or running.    -   Muscle contractures of Achilles tendon and hamstrings impair        functionality because the muscle fibers shorten and fibrose in        connective tissue    -   Progressive difficulty walking    -   Muscle fiber deformities    -   Pseudohypertrophy (enlarging) of tongue and calf muscles. The        muscle tissue is eventually replaced by fat and connective        tissue, hence the term pseudohypertrophy.    -   Higher risk of neurobehavioral disorders (e.g., ADHD), learning        disorders (dyslexia), and non-progressive weaknesses in specific        cognitive skills (in particular short-term verbal memory), which        are believed to be the result of absent or dysfunctional        dystrophin in the brain.    -   Eventual loss of ability to walk (usually by the age of 12)    -   Skeletal deformities (including scoliosis in some cases)    -   Trouble getting up from lying or sitting position        The condition can often be observed clinically from the moment        the patient takes his first steps, and the ability to walk        usually completely disintegrates between the time the boy is 9        to 12 years of age. Most men affected with DMD become        essentially “paralyzed from the neck down” by the age of 21.        Muscle wasting begins in the legs and pelvis, then progresses to        the muscles of the shoulders and neck, followed by loss of arm        muscles and respiratory muscles. Calf muscle enlargement        (pseudohypertrophy) is quite obvious. Cardiomyopathy        particularly (dilated cardiomyopathy) is common, but the        development of congestive heart failure or arrhythmia (irregular        heartbeat) is only occasional.

A positive Gowers' sign reflects the more severe impairment of the lowerextremities muscles. The child helps himself to get up with upperextremities: first by rising to stand on his arms and knees, and then“walking” his hands up his legs to stand upright. Affected childrenusually tire more easily and have less overall strength than theirpeers. Creatine kinase (CPK-MM) levels in the bloodstream are extremelyhigh. An electromyography (EMG) shows that weakness is caused bydestruction of muscle tissue rather than by damage to nerves. Genetictesting can reveal genetic errors in the Xp21 gene. A muscle biopsy(immunohistochemistry or immunoblotting) or genetic test (blood test)confirms the absence of dystrophin, although improvements in genetictesting often make this unnecessary.

-   -   Abnormal heart muscle (cardiomyopathy)    -   Congestive heart failure or irregular heart rhythm (arrhythmia)    -   Deformities of the chest and back (scoliosis)    -   Enlarged muscles of the calves, buttocks, and shoulders (around        age 4 or 5). These muscles are eventually replaced by fat and        connective tissue (pseudohypertrophy).    -   Loss of muscle mass (atrophy)    -   Muscle contractures in the heels, legs    -   Muscle deformities    -   Respiratory disorders, including pneumonia and swallowing with        food or fluid passing into the lungs (in late stages of the        disease)

C. Causes

Duchenne muscular dystrophy (DMD) is caused by a mutation of thedystrophin gene at locus Xp21, located on the short arm of the Xchromosome. Dystrophin is responsible for connecting the cytoskeleton ofeach muscle fiber to the underlying basal lamina (extracellular matrix),through a protein complex containing many subunits. The absence ofdystrophin permits excess calcium to penetrate the sarcolemma (the cellmembrane). Alterations in calcium and signaling pathways cause water toenter into the mitochondria, which then burst.

In skeletal muscle dystrophy, mitochondrial dysfunction gives rise to anamplification of stress-induced cytosolic calcium signals and anamplification of stress-induced reactive-oxygen species (ROS)production. In a complex cascading process that involves severalpathways and is not clearly understood, increased oxidative stresswithin the cell damages the sarcolemma and eventually results in thedeath of the cell. Muscle fibers undergo necrosis and are ultimatelyreplaced with adipose and connective tissue.

DMD is inherited in an X-linked recessive pattern. Females willtypically be carriers for the disease while males will be affected.Typically, a female carrier will be unaware they carry a mutation untilthey have an affected son. The son of a carrier mother has a 50% chanceof inheriting the defective gene from his mother. The daughter of acarrier mother has a 50% chance of being a carrier and a 50% chance ofhaving two normal copies of the gene. In all cases, an unaffected fatherwill either pass a normal Y to his son or a normal X to his daughter.Female carriers of an X-linked recessive condition, such as DMD, canshow symptoms depending on their pattern of X-inactivation.

Duchenne muscular dystrophy has an incidence of 1 in 5000 male infants.Mutations within the dystrophin gene can either be inherited or occurspontaneously during germline transmission. A table of exemplary butnon-limiting mutations and corresponding models are set forth below:

Deletion, small insertion and nonsense mutations Name of Mouse ModelExon 44 ΔEx44 Exon 52 ΔEx52 Exon 43 ΔEx43

D. Diagnosis

Genetic counseling is advised for people with a family history of thedisorder. Duchenne muscular dystrophy can be detected with about 95%accuracy by genetic studies.

DNA Test.

The muscle-specific isoform of the dystrophin gene is composed of 79exons, and DNA testing and analysis can usually identify the specifictype of mutation of the exon or exons that are affected. DNA testingconfirms the diagnosis in most cases.

Muscle Biopsy.

If DNA testing fails to find the mutation, a muscle biopsy test may beperformed. A small sample of muscle tissue is extracted (usually with ascalpel instead of a needle) and a dye is applied that reveals thepresence of dystrophin. Complete absence of the protein indicates thecondition.

Over the past several years DNA tests have been developed that detectmore of the many mutations that cause the condition, and muscle biopsyis not required as often to confirm the presence of Duchenne's.

Prenatal Tests.

DMD is carried by an X-linked recessive gene. Males have only one Xchromosome, so one copy of the mutated gene will cause DMD. Fatherscannot pass X-linked traits on to their sons, so the mutation istransmitted by the mother.

If the mother is a carrier, and therefore one of her two X chromosomeshas a DMD mutation, there is a 50% chance that a female child willinherit that mutation as one of her two X chromosomes, and be a carrier.There is a 50% chance that a male child will inherit that mutation ashis one X chromosome, and therefore have DMD.

Prenatal tests can tell whether their unborn child has the most commonmutations. There are many mutations responsible for DMD, and some havenot been identified, so genetic testing only works when family memberswith DMD have a mutation that has been identified.

Prior to invasive testing, determination of the fetal sex is important;while males are sometimes affected by this X-linked disease, female DMDis extremely rare. This can be achieved by ultrasound scan at 16 weeksor more recently by free fetal DNA testing. Chorion villus sampling(CVS) can be done at 11-14 weeks, and has a 1% risk of miscarriage.Amniocentesis can be done after 15 weeks, and has a 0.5% risk ofmiscarriage. Fetal blood sampling can be done at about 18 weeks. Anotheroption in the case of unclear genetic test results is fetal musclebiopsy.

E. Treatment

There is no current cure for DMD, and an ongoing medical need has beenrecognized by regulatory authorities. Phase 1-2a trials with exonskipping treatment for certain mutations have halted decline andproduced clinical improvements in walking. Sarepta's drug Exondys 51(eteplirsen) has recently received FDA approval. However, treatment isgenerally aimed at controlling the onset of symptoms to maximize thequality of life, and include the following:

-   -   Corticosteroids such as prednisolone and deflazacort increase        energy and strength and defer severity of some symptoms.    -   Randomized control trials have shown that beta-2-agonists        increase muscle strength but do not modify disease progression.        Follow-up time for most RCTs on beta2-agonists is only around 12        months and hence results cannot be extrapolated beyond that time        frame.    -   Mild, non-jarring physical activity such as swimming is        encouraged. Inactivity (such as bed rest) can worsen the muscle        disease.    -   Physical therapy is helpful to maintain muscle strength,        flexibility, and function.    -   Orthopedic appliances (such as braces and wheelchairs) may        improve mobility and the ability for self-care. Form-fitting        removable leg braces that hold the ankle in place during sleep        can defer the onset of contractures.    -   Appropriate respiratory support as the disease progresses is        important.        Comprehensive multi-disciplinary care standards/guidelines for        DMD have been developed by the Centers for Disease Control and        Prevention (CDC), and were published in two parts in The Lancet        Neurology in 2010. To download the two articles in PDF format,        go to the TREAT-NMD website.

1. Physical Therapy

Physical therapists are concerned with enabling patients to reach theirmaximum physical potential. Their aim is to:

-   -   minimize the development of contractures and deformity by        developing a programme of stretches and exercises where        appropriate    -   anticipate and minimize other secondary complications of a        physical nature by recommending bracing and durable medical        equipment    -   monitor respiratory function and advise on techniques to assist        with breathing exercises and methods of clearing secretions

2. Respiration Assistance

Modern “volume ventilators/respirators,” which deliver an adjustablevolume (amount) of air to the person with each breath, are valuable inthe treatment of people with muscular dystrophy related respiratoryproblems. The ventilator may require an invasive endotracheal ortracheotomy tube through which air is directly delivered, but, for somepeople non-invasive delivery through a face mask or mouthpiece issufficient. Positive airway pressure machines, particularly bi-levelones, are sometimes used in this latter way. The respiratory equipmentmay easily fit on a ventilator tray on the bottom or back of a powerwheelchair with an external battery for portability.

Ventilator treatment may start in the mid to late teens when therespiratory muscles can begin to collapse. If the vital capacity hasdropped below 40% of normal, a volume ventilator/respirator may be usedduring sleeping hours, a time when the person is most likely to be underventilating (“hypoventilating”). Hypoventilation during sleep isdetermined by a thorough history of sleep disorder with an oximetrystudy and a capillary blood gas (See Pulmonary Function Testing). Acough assist device can help with excess mucus in lungs byhyperinflation of the lungs with positive air pressure, then negativepressure to get the mucus up. If the vital capacity continues to declineto less than 30 percent of normal, a volume ventilator/respirator mayalso be needed during the day for more assistance. The person graduallywill increase the amount of time using the ventilator/respirator duringthe day as needed.

F. Prognosis

Duchenne muscular dystrophy is a progressive disease which eventuallyaffects all voluntary muscles and involves the heart and breathingmuscles in later stages. The life expectancy is currently estimated tobe around 25, but this varies from patient to patient. Recentadvancements in medicine are extending the lives of those afflicted. TheMuscular Dystrophy Campaign, which is a leading UK charity focusing onall muscle disease, states that “with high standards of medical careyoung men with Duchenne muscular dystrophy are often living well intotheir 30s.”

In rare cases, persons with DMD have been seen to survive into theforties or early fifties, with the use of proper positioning inwheelchairs and beds, ventilator support (via tracheostomy ormouthpiece), airway clearance, and heart medications, if required. Earlyplanning of the required supports for later-life care has shown greaterlongevity in people living with DMD.

Curiously, in the mdx mouse model of Duchenne muscular dystrophy, thelack of dystrophin is associated with increased calcium levels andskeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) areprotected and do not undergo myonecrosis. ILM have a calcium regulationsystem profile suggestive of a better ability to handle calcium changesin comparison to other muscles, and this may provide a mechanisticinsight for their unique pathophysiological properties. The ILM mayfacilitate the development of novel strategies for the prevention andtreatment of muscle wasting in a variety of clinical scenarios.

II. CRISPR SYSTEMS

A. CRISPR and Nucleases

CRISPRs (clustered regularly interspaced short palindromic repeats) areDNA loci containing short repetitions of base sequences. Each repetitionis followed by short segments of “spacer DNA” from previous exposures toa virus. CRISPRs are found in approximately 40% of sequenced eubacteriagenomes and 90% of sequenced archaea. CRISPRs are often associated withCas genes that code for proteins related to CRISPRs. The CRISPR/Cassystem is a prokaryotic immune system that confers resistance to foreigngenetic elements such as plasmids and phages and provides a form ofacquired immunity. CRISPR spacers recognize and silence these exogenousgenetic elements like RNAi in eukaryotic organisms.

Repeats were first described in 1987 for the bacterium Escherichia coli.In 2000, similar clustered repeats were identified in additionalbacteria and archaea and were termed Short Regularly Spaced Repeats(SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encodingputative nuclease or helicase proteins, were found to be associated withCRISPR repeats (the cas, or CRISPR-associated genes).

In 2005, three independent researchers showed that CRISPR spacers showedhomology to several phage DNA and extrachromosomal DNA such as plasmids.This was an indication that the CRISPR/cas system could have a role inadaptive immunity in bacteria. Koonin and colleagues proposed thatspacers serve as a template for RNA molecules, analogously to eukaryoticcells that use a system called RNA interference.

In 2007 Barrangou, Horvath (food industry scientists at Danisco) andothers showed that they could alter the resistance of Streptococcusthermophilus to phage attack with spacer DNA. Doudna and Charpentier hadindependently been exploring CRISPR-associated proteins to learn howbacteria deploy spacers in their immune defenses. They jointly studied asimpler CRISPR system that relies on a protein called Cas9. They foundthat bacteria respond to an invading phage by transcribing spacers andpalindromic DNA into a long RNA molecule that the cell then usestracrRNA and Cas9 to cut it into pieces called crRNAs.

CRISPR was first shown to work as a genome engineering/editing tool inhuman cell culture by 2012 It has since been used in a wide range oforganisms including baker's yeast (S. cerevisiae), zebra fish, nematodes(C. elegans), plants, mice, and several other organisms. AdditionallyCRISPR has been modified to make programmable transcription factors thatallow scientists to target and activate or silence specific genes.Libraries of tens of thousands of guide RNAs are now available.

The first evidence that CRISPR can reverse disease symptoms in livinganimals was demonstrated in March 2014, when MIT researchers cured miceof a rare liver disorder. Since 2012, the CRISPR/Cas system has beenused for gene editing (silencing, enhancing or changing specific genes)that even works in eukaryotes like mice and primates. By inserting aplasmid containing cas genes and specifically designed CRISPRs, anorganism's genome can be cut at any desired location.

CRISPR repeats range in size from 24 to 48 base pairs. They usually showsome dyad symmetry, implying the formation of a secondary structure suchas a hairpin, but are not truly palindromic. Repeats are separated byspacers of similar length. Some CRISPR spacer sequences exactly matchsequences from plasmids and phages, although some spacers match theprokaryote's genome (self-targeting spacers). New spacers can be addedrapidly in response to phage infection.

CRISPR-associated (cas) genes are often associated with CRISPRrepeat-spacer arrays. As of 2013, more than forty different Cas proteinfamilies had been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

Exogenous DNA is apparently processed by proteins encoded by Cas genesinto small elements (˜30 base pairs in length), which are then somehowinserted into the CRISPR locus near the leader sequence. RNAs from theCRISPR loci are constitutively expressed and are processed by Casproteins to small RNAs composed of individual, exogenously-derivedsequence elements with a flanking repeat sequence. The RNAs guide otherCas proteins to silence exogenous genetic elements at the RNA or DNAlevel. Evidence suggests functional diversity among CRISPR subtypes. TheCse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. In other prokaryotes, Cas6processes the CRISPR transcripts. Interestingly, CRISPR-based phageinactivation in E. coli requires Cascade and Cas3, but not Cas1 andCas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosusand other prokaryotes form a functional complex with small CRISPR RNAsthat recognizes and cleaves complementary target RNAs. RNA-guided CRISPRenzymes are classified as type V restriction enzymes.

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with twoactive cutting sites, one for each strand of the double helix. The teamdemonstrated that they could disable one or both sites while preservingCas9's ability to home located its target DNA. Jinek et al. (2012)combined tracrRNA and spacer RNA into a “single-guide RNA” moleculethat, mixed with Cas9, could find and cut the correct DNA targets. Jineket al. (2012) proposed that such synthetic guide RNAs might be able tobe used for gene editing.

Cas9 proteins are highly enriched in pathogenic and commensal bacteria.CRISPR/Cas-mediated gene regulation may contribute to the regulation ofendogenous bacterial genes, particularly during bacterial interactionwith eukaryotic hosts. For example, Cas protein Cas9 of Francisellanovicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) torepress an endogenous transcript encoding a bacterial lipoprotein thatis critical for F. novicida to dampen host response and promotevirulence. Wang et al. (2013) showed that coinjection of Cas9 mRNA andsgRNAs into the germline (zygotes) generated nice with mutations.Delivery of Cas9 DNA sequences also is contemplated.

See also U.S. Patent Publication 2014/0068797, which is incorporated byreference in its entirety.

Clustered Regularly Interspaced Short Palindromic Repeats fromPrevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technologyanalogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonucleaseof a class II CRISPR/Cas system. This acquired immune mechanism is foundin Prevotella and Francisella bacteria. It prevents genetic damage fromviruses. Cpf1 genes are associated with the CRISPR locus, coding for anendonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 isa smaller and simpler endonuclease than Cas9, overcoming some of theCRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multipleapplications, including treatment of genetic illnesses and degenerativeconditions.

CRISPR/Cpf1 was found by searching a published database of bacterialgenetic sequences for promising bits of DNA. Its identification throughbioinformatics as a CRISPR system protein, its naming, and a hiddenMarkov model (HMM) for its detection were provided in 2012 in a releaseof the TIGRFAMs database of protein families. Cpf1 appears in manybacterial species. The ultimate Cpf1 endonuclease that was developedinto a tool for genome editing was taken from one of the first 16species known to harbor it. Two candidate enzymes from Acidaminococcusand Lachnospiraceae display efficient genome-editing activity in humancells.

A smaller version of Cas9 from the bacterium Staphylococcus aureus is apotential alternative to Cpf1.

The systems CRISPR/Cas are separated into three classes. Class 1 usesseveral Cas proteins together with the CRISPR RNAs (crRNA) to build afunctional endonuclease. Class 2 CRISPR systems use a single Cas proteinwith a crRNA. Cpf1 has been recently identified as a Class II, Type VCRISPR/Cas systems containing a 1,300 amino acid protein.

The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed bya helical region, a RuvC-II and a zinc finger-like domain. The Cpf1protein has a RuvC-like endonuclease domain that is similar to the RuvCdomain of Cas9. Furthermore, Cpf1 does not have a HNH endonucleasedomain, and the N-terminal of Cpf1 does not have the alpha-helicalrecognition lobe of Cas9.

Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionallyunique, being classified as Class 2, type V CRISPR system. The Cpf1 lociencode Cas1, Cas2 and Cas4 proteins more similar to types I and III thanfrom type II systems. Database searches suggest the abundance ofCpf1-family proteins in many bacterial species.

Functional Cpf1 doesn't need the tracrRNA, therefore, only crRNA isrequired. This benefits genome editing because Cpf1 is not only smallerthan Cas9, but also it has a smaller sgRNA molecule (proximately half asmany nucleotides as Cas9).

The Cpf1-crRNA complex cleaves target DNA or RNA by identification of aprotospacer adjacent motif 5′-YTN-3′ (where “Y” is a pyrimidine and “N”is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM targetedby Cas9. After identification of PAM, Cpf1 introduces a sticky-end-likeDNA double-stranded break of 4 or 5 nucleotides overhang.

The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA thatfinds and positions the complex at the correct spot on the double helixto cleave target DNA. CRISPR/Cpf1 systems activity has three stages:

-   -   Adaptation, during which Cas1 and Cas2 proteins facilitate the        adaptation of small fragments of DNA into the CRISPR array;    -   Formation of crRNAs: processing of pre-cr-RNAs producing of        mature crRNAs to guide the Cas protein; and    -   Interference, in which the Cpf1 is bound to a crRNA to form a        binary complex to identify and cleave a target DNA sequence.

B. sgRNA

As an RNA guided protein, Cas9 requires a short RNA to direct therecognition of DNA targets (Mali et al., 2013a). Though Cas9preferentially interrogates DNA sequences containing a PAM sequence NGGit can bind here without a protospacer target. However, the Cas9-sgRNAcomplex requires a close match to the sgRNA to create a double strandbreak (Cho et al., 2013; Hsu et al., 2013). CRISPR sequences in bacteriaare expressed in multiple RNAs and then processed to create guidestrands for RNA (Bikard et al., 2013). Because Eukaryotic systems lacksome of the proteins required to process CRISPR RNAs the syntheticconstruct sgRNA was created to combine the essential pieces of RNA forCas9 targeting into a single RNA expressed with the RNA polymerase typeIII promoter U6 (Mali et al., 2013b,c). Synthetic sgRNAs are slightlyover 100 bp at the minimum length and contain a portion which is targetsthe 20 protospacer nucleotides immediately preceding the PAM sequenceNGG; sgRNAs do not contain a PAM sequence.

III. NUCLEIC ACID DELIVERY

As discussed above, in certain embodiments, expression cassettes areemployed to express a transcription factor product, either forsubsequent purification and delivery to a cell/subject, or for usedirectly in a genetic-based delivery approach. Expression requires thatappropriate signals be provided in the vectors, and include variousregulatory elements such as enhancers/promoters from both viral andmammalian sources that drive expression of the genes of interest incells. Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined. The conditions for theuse of a number of dominant drug selection markers for establishingpermanent, stable cell clones expressing the products are also provided,as is an element that links expression of the drug selection markers toexpression of the polypeptide.

A. Regulatory Elements

Throughout this application, the term “expression cassette” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed and translated, i.e., is underthe control of a promoter. A “promoter” refers to a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene. The phrase “under transcriptional control” means that thepromoter is in the correct location and orientation in relation to thenucleic acid to control RNA polymerase initiation and expression of thegene. An “expression vector” is meant to include expression cassettescomprised in a genetic construct that is capable of replication, andthus including one or more of origins of replication, transcriptiontermination signals, poly-A regions, selectable markers, andmultipurpose cloning sites.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

In certain embodiments, viral promoters such as the humancytomegalovirus (CMV) immediate early gene promoter, the SV40 earlypromoter, the Rous sarcoma virus long terminal repeat, rat insulinpromoter and glyceraldehyde-3-phosphate dehydrogenase can be used toobtain high-level expression of the coding sequence of interest. The useof other viral or mammalian cellular or bacterial phage promoters whichare well-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized. Further, selection of a promoter that is regulated inresponse to specific physiologic signals can permit inducible expressionof the gene product.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters/enhancers and inducible promoters/enhancersthat could be used in combination with the nucleic acid encoding a geneof interest in an expression construct. Additionally, anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of the gene. Eukaryoticcells can support cytoplasmic transcription from certain bacterialpromoters if the appropriate bacterial polymerase is provided, either aspart of the delivery complex or as an additional genetic expressionconstruct.

TABLE A Promoter and/or Enhancer Promoter/ Enhancer ReferencesImmunoglobulin Banerji et al., 1983; Gilles et al., 1983; GrosschedlHeavy Chain et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Queen and Baltimore, 1983; Picard et al., 1984 LightChain T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondoet al.; 1990 HLA DQ a and/or Sullivan et al., 1987 DQ β β-InterferonGoodbourn et al., 1986; Fujita et al., 1987; Goodbourn and Maniatis etal., 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Greene et al.,1989; Lin et al., 1990 Receptor MHC Class II 5 Koch et al., 1989 MHCClass II Sherman et al., 1989 HLA-DRa β-Actin Kawamoto et al., 1988; Nget al.; 1989 Muscle Creatine Jaynes et al., 1988; Horlick et al., 1989;Johnson Kinase (MCK) et al., 1989 Prealbumin Costa et al., 1988(Transthyretin) Elastase I Ornitz et al., 1987 Metallothionein Karin etal., 1987; Culotta et al., 1989 (MTII) Collagenase Pinkert et al., 1987;Angel et al., 1987a Albumin Pinkert et al., 1987; Tronche et al., 1989,1990 α-Fetoprotein Godbout et al., 1988; Campere and Tilghman et al.,1989 t-Globin Bodine and Ley et al., 1987; Perez-Stable et al., 1990β-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras Triesman,1986; Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural CellHirsh et al., 1990 Adhesion Molecule (NCAM) α₁-Antitrypain Latimer etal., 1990 H2B (TH2B) Hwang et al., 1990 Histone Mouse and/or Ripe etal., 1989 Type I Collagen Glucose-Regulated Chang et al., 1989 Proteins(GRP94 and GRP78) Rat Growth Larsen et al., 1986 Hormone Human SerumEdbrooke et al., 1989 Amyloid A (SAA) Troponin I (TN I) Yutzey et al.,1989 Platelet-Derived Pech et al., 1989 Growth Factor (PDGF) DuchenneMuscular Klamut et al., 1990 Dystrophy SV40 Banerji et al., 1981; Moreauet al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr and Clarkeet al., 1986; Imbra and Karin et al., 1986; Kadesch and Berg, 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinsonet al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et al.,1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla and Siddiqui et al., 1986; Jameel and Siddiqui,1986; Shaul and Ben-Levy, 1987; Spandau et al., 1988; Vannice et al.,1988 Human Muesing et al., 1987; Hauber and Cullen et al.,Immunodeficiency 1988; Jakobovits et al., 1988; Feng et al., 1988; VirusTakebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspiaet al., 1989; Sharp et al., 1989; Braddock et al., 1989 CytomegalovirusWeber et al., 1984; Boshart et al., 1985; Foecking (CMV) et al., 1986Gibbon Ape Holbrook et al., 1987; Quinn et al., 1989 Leukemia Virus

TABLE B Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982a, b; Heavy metals Haslinger et al.,1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987,Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse Glucocorticoids Huang et al., 1981; Lee mammary et al., 1981;Majors and tumor virus) Varmas et al., 1983; Chandler et al., 1983;Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernieret al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, Hug et al., 1988 Newcastle DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I GeneInterferon Blanar et al., 1989 H-2κb HSP70 ElA, SV40 Large Taylor etal., 1989, 1990a, T Antigen 1990b Proliferin Phorbol Ester-TPA Mordacqand Linzer, 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidThyroid Hormone Chatterjee et al., 1989 Stimulating Hormone α Gene

Of particular interest are muscle specific promoters. These include themyosin light chain-2 promoter (Franz et al., 1994; Kelly et al., 1995),the muscle creatine kinase enhancer (Jaynes et al., 1988; Horlick etal., 1989; Johnson et al., 1989), the α-actin promoter (Moss et al.,1996), the troponin 1 promoter (Bhaysar et al., 1996); the Na⁺/Ca²⁺exchanger promoter (Barnes et al., 1997), the dystrophin promoter(Kimura et al., 1997), the α7 integrin promoter (Ziober and Kramer,1996), the brain natriuretic peptide promoter (LaPointe et al., 1996)and the αB-crystallin/small heat shock protein promoter(Gopal-Srivastava, 1995), α-myosin heavy chain promoter(Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al.,1988).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

B. 2A Protease

In some embodiments, a 2A-like self-cleaving domain from the insectvirus Thosea asigna (TaV 2A peptide) (Chang et al., 2009) is used. These2A-like domains have been shown to function across eukaryotes and causecleavage of amino acids to occur co-translationally within the 2A-likepeptide domain. Therefore, inclusion of TaV 2A peptide allows theexpression of multiple proteins from a single mRNA transcript.Importantly, the domain of TaV when tested in eukaryotic systems haveshown greater than 99% cleavage activity (Donnelly et al., 2001).

C. Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In one system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Animal studies suggestedthat recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection. A reagent known as Lipofectamine 2000™ iswidely used and commercially available.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). A syntheticneoglycoprotein, which recognizes the same receptor as ASOR, has beenused as a gene delivery vehicle (Ferkol et al., 1993; Perales et al.,1994) and epidermal growth factor (EGF) has also been used to delivergenes to squamous carcinoma cells (Myers, EP 0273085).

IV. METHODS OF MAKING TRANSGENIC MICE

A particular embodiment of the present invention provides transgenicanimals that contain mutations in the dystrophin gene. Also, transgenicanimals may express a marker that reflects the production of mutant ornormal dystrophin gene product.

In a general aspect, a transgenic animal is produced by the integrationof a given construct into the genome in a manner that permits theexpression of the transgene using methods discussed above. Methods forproducing transgenic animals are generally described by Wagner and Hoppe(U.S. Pat. No. 4,873,191; incorporated herein by reference), andBrinster et al. (1985; incorporated herein by reference).

Typically, the construct is transferred by microinjection into afertilized egg. The microinjected eggs are implanted into a host female,and the progeny are screened for the expression of the transgene.Transgenic animals may be produced from the fertilized eggs from anumber of animals including, but not limited to reptiles, amphibians,birds, mammals, and fish.

RNA for microinjection can be prepared by any means known in the art.For example, RNA for microinjection can be cleaved with enzymesappropriate for removing the bacterial plasmid sequences, and the RNAfragments electrophoresed on 1% agarose gels in TBE buffer, usingstandard techniques. The RNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. RNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The RNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D® column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind RNA to the column matrix. After one wash with3 ml of low salt buffer, the RNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. RNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA. Other methods for purification of RNA formicroinjection are described in Palmiter et al. (1982a,b); and inSambrook and Russell (2001).

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby CO₂ asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.C57BL/6 or Swiss mice or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmakers forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

V. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender drugs, proteins or delivery vectors stable and allow for uptakeby target cells. Aqueous compositions of the present invention comprisean effective amount of the drug, vector or proteins, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includessolvents, buffers, solutions, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike acceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route, but generallyincluding systemic administration. This includes oral, nasal, or buccal.Alternatively, administration may be by intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection, or by directinjection into muscle tissue. Such compositions would normally beadministered as pharmaceutically acceptable compositions, as describedsupra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

VI. SEQUENCE TABLES

TABLE 1 Sequence of primers for sgRNA targeting DMDand Dmd exon 45 splicing acceptor site SEQ ID ID Sequence (5′-3′) NO.Ex45-gRNA#3-Top CACCGCGCTGCCCAATGCCATCCTG 1 Ex45-gRNA#3-BotAAACCAGGATGGCATTGGGCAGCGC 2 Ex45-gRNA#4-Top CACCGCTTACAGGAACTCCAGGA 3Ex45-gRNA#4-Bot AAACTCCTGGAGTTCCTGTAAGC 4 Ex45-gRNA#5-TopCACCGAGGAACTCCAGGATGGCATT 5 Ex45-gRNA#5-Bot AAACAATGCCATCCTGGAGTTCCTC 6Ex45-gRNA#6-Top CACCGCGCTGCCCAATGCCATCC 7 Ex45-gRNA#6-BotAAACGGATGGCATTGGGCAGCGC 8 mDmd-T7E1-Ex45-F CTAACATAAAAGGTGTCTTTCTATC 9mDmd-T7E1-Ex45-R GGCAATCCCTCATGATTTTTAGCAC 10 DMD-T7E1-Ex45-FGTCTTTCTGTCTTGTATCCTTTGG 11 DMD-T7E1-Ex45-R AATGTTAGTGCCTTTCACCC 12

TABLE 2 Sequence of primers for sgRNA targeting Dmdexon 44 to generate the mouse model SEQ  Mouse NO. ID ModelSequence (5′-3′) ID mDmd-In44-2-Top Δex44 CACCGGTAGTTCTGAATCAG 13 GAGGAmDmd-In44-2-Bot Δex44 AAACTCCTCCTGATTCAGAA 14 CTACC mDmd-In44-6-TopΔex44 CACCGTATGTTGGAACCAGT 15 CCAGA mDmd-In44-6-Bot Δex44AAACTCTGGACTGGTTCCAA 16 CATAC

TABLE 3 Sequence of primers for in vitro transcription of sgRNA SEQMouse ID ID Model Sequence (5′-3′) NO exon 44_T7-In44-2-F Δex44GAATTGTAATACGACTC 17 ACTATAGGGGTAGTTCT GAATCAGGAGGA exon 44_T7-In44-6-FΔex44 GAATTGTAATACGACTC 18 ACTATAGGGTATGTTGG AACCAGTCCAGA exon 44_T7-RvΔex44 AAAAGCACCGACTCGGT 19 GCCAC

TABLE 4 Sequence of primers for genotyping SEQ Mouse ID ID ModelSequence (5′-3′) NO. Geno44-F Δex44 GCTGAGGGGGAGACAGTAGA 20 Geno44-RΔex44 TCAGAAGGCATTTTGTCAAT 21

TABLE 5 gRNA genomic target sequences SEQ ID sgRNA ID Sequence (5′-3′)NO. Ex45-gRNA#3 CGCTGCCCAATGCCATCCTG 39 Ex45-gRNA#4 ATCTTACAGGAACTCCAGGA40 Ex45-gRNA#5 AGGAACTCCAGGATGGCATT 41 Ex45-gRNA#6 CGCTGCCCAATGCCATCC 42Ex43-gRNA#1 GTTTTAAAATTTTTATATTA 43 Ex43-gRNA#2 TTTTATATTACAGAATATAA 44Ex43-gRNA#4 TATGTGTTACCTACCCTTGT 45 Ex43-gRNA#6 GTACAAGGACCGACAAGGGT 46

TABLE 6 gRNA sequences SEQ ID sgRNA ID Sequence (5′-3′) NO. Ex45-gRNA#3CAGGAUGGCAUUGGGCAGCG 47 Ex45-gRNA#4 UCCUGGAGUUCCUGUAAGAU 48 Ex45-gRNA#5AAUGCCAUCCUGGAGUUCCU 49 Ex45-gRNA#6 GGAUGGCAUUGGGCAGCG 50 Ex43-gRNA#1UAAUAUAAAAAUUUUAAAAC 51 Ex43-gRNA#2 UUAUAUUCUGUAAUAUAAAA 52 Ex43-gRNA#4ACAAGGGUAGGUAACACAUA 53 Ex43-gRNA#6 ACCCUUGUCGGUCCUUGUAC 54

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques which function well in the practice of the disclosure, andthus can be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

Example 1—Materials and Methods

Study Approval.

All experimental procedures involving animals in this study werereviewed and approved by the University of Texas Southwestern MedicalCenter's Institutional Animal Care and Use Committee.

Plasmids.

The pSpCas9(BB)-2A-GFP (PX458) plasmid containing the human codonoptimized SpCas9 gene with 2A-EGFP and the backbone of sgRNA waspurchased from Addgene (Plasmid #48138). Cloning of sgRNA was done usingBbs I sites. The AAV TRISPR-CK8-GFP plasmid containing three sgRNAsdriven by U6, H1 or 7SK promoter and GFP driven by CK8 promoter.

Human iPSCs Maintenance and Nucleofection.

Human iPSCs were cultured in mTeSR™1 media (STEMCELL Technologies) andpassaged approximately every 4 days (1:18 split ratio). One hour beforenucleofection, iPSCs were treated with 10 μM ROCK inhibitor (Y-27632)and dissociated into single cells using Accutase (Innovative CellTechnologies, Inc.). 1×10⁶ iPS cells were mixed with 5 μg ofpLbCpf1-2A-GFP or pAsCpf1-2A-GFP plasmid and nucleofected using the P3Primary Cell 4D-Nucleofector X kit (Lonza) according to manufacturer'sprotocol. After nucleofection, iPSCs were cultured in mTeSR™ 1 mediasupplemented with 10 μM ROCK inhibitor, penicillin-streptomycin (1:100)(ThermoFisher Scientific) and 100 μg/ml Primosin (InvivoGen). Three dayspost-nucleofection, GFP(+) and (−) cells were sorted by FACS andsubjected to T7E1 assay. Single clones derived from GFP(+) iPSCs werepicked and sequenced.

Genomic DNA Isolation.

Genomic DNA of mouse 10T1/2 fibroblasts, mouse C2C12, human 293 andhuman iPSCs was isolated using DirectPCR (cell) lysis reagent (VIAGEN)according to manufacturer's protocol.

PCR Amplification of Genomic DNA.

Genomic DNA was PCR-amplified using GoTaq DNA polymerase (Promega) withprimers. PCR products were gel purified and subcloned into pCRII-TOPOvector (Invitrogen) according to the manufacturer's protocol. Individualclones were picked and the DNA was sequenced. Primer sequences arelisted in supplemental material.

T7E1 Analysis of PCR Products.

Mismatched duplex DNA was obtained by denaturing/renaturing of 25 μl ofthe genomic PCR product using the following conditions: 95° C. for 5mins, 95° C. to 85° C. (−2.0° C./seconds), 85° C. to 25° C. (−0.1°C./seconds), hold at 4° C. Then 25 μl of the mismatched duplex DNA wasincubated with 2.7 μl of 10×NEB buffer 2 and 0.3 μl of T7E1 (New EnglandBioLabs) at 37° C. for 90 minutes. The T7E1 digested PCR product wasanalyzed by 2% agarose gel electrophoresis.

Human Cardiomyocyte Differentiation.

Human iPSCs were cultured in mTeSR™1 media for 3 days until they reached90% confluence. To differentiate the iPSCs to cardiomyocytes, the iPSCswere cultured in CDM3-C media for 2 days, followed by CDM3-59 media for2 days, followed by CDM3 media for 6 days, followed by selective mediafor 10 days and lastly by basal media for 2 days. Then, thecardiomyocytes were dissociated using TrypLE media and re-plated at2×10⁶ per well in a 6-well dish. Differentiation medium recipe can befound in supplemental materials.

Dystrophin Western Blot Analysis.

After 30 days post-differentiation, 2×10⁶ cardiomyocytes were harvestedand lysed with lysis buffer (10% SDS, 62.5 mM Tris pH=6.8, 1 mM EDTA,and protease inhibitor). Cell lysates were passed through a 22 G syringeand then a 27 G syringe, 10 times each. Protein concentration wasdetermined by BCA assay and 50 ug of total protein was loaded onto anacrylamide gel. After running at 100V (20 mA) for 5 hours and followedby 1 hour 20 min transfer to PVDF membrane at 35V (200 mA) at 4° C. Theblot was incubated with mouse anti-dystrophin antibody (MANDYS8,Sigma-Aldrich, D8168) at 4° C. overnight and with goat anti-mouse HRPantibody (Bio-Rad Laboratories) at RT for 1 hour. The blot was developedusing Western Blotting Luminol Reagent (Santa Cruz, sc-2048). Theloading control was determined by blotting with mouse anti-vinculinantibody (Sigma-Aldrich, V9131).

CRISPR/Cas9-Mediated Exon 44 Deletion in Mice.

Two single-guide RNA (sgRNA) specific intronic regions surrounding exon44 sequence of the mouse Dmd locus were cloned into vector PX458 usingthe primers from Table 1. For the in vitro transcription of sgRNA, T7promoter sequence was added to the sgRNA template by PCR using theprimers from Table 2. The gel purified PCR products were used astemplate for in vitro transcription using the MEGAshortscript T7 Kit(Life Technologies). sgRNA were purified by MEGAclear kit (LifeTechnologies) and eluted with nuclease-free water (Ambion). Theconcentration of guide RNA was measured by a NanoDrop instrument (ThermoScientific).

Genotyping of ΔEx44 Mice.

ΔEx44 mice were genotyped using primers encompassing the targeted regionfrom Table 3. Tail biopsies were digested in 100 μL of 25 mM NaOH, 0.2mM EDTA (pH 12) for 20 min at 95° C. Tails were briefly centrifugedfollowed by addition of 100 μL of 40 mM Tris.HCl (pH 5) and mixed tohomogenize. Two microliters of this reaction was used for subsequent PCRreactions with the primers below, followed by gel electrophoresis.

Histological Analysis of Muscles.

Skeletal muscles from WT and ΔEx44 mice were individually dissected andcryoembedded in a 1:2 volume mixture of Gum Tragacanth powder(Sigma-Aldrich) to Tissue Freezing Medium (TFM) (Triangle Bioscience).All embeds were snap frozen in isopentane heat extractant supercooled to−155° C. Resulting blocks were stored overnight at −80° C. prior tosectioning. Eight-micron transverse sections of skeletal muscle, andfrontal sections of heart were prepared on a Leica CM3050 cryostat andair-dried prior to same day staining. H&E staining was performedaccording to established staining protocols and dystrophinimmunohistochemistry was performed using MANDYS8 monoclonal antibody(Sigma-Aldrich) with modifications to manufacturer's instructions. Inbrief, cryostat sections were thawed and rehydrated/delipidated in 1%triton/phosphate-buffered-saline, pH 7.4 (PBS). Following delipidation,sections were washed free of Triton, incubated with mouse IgG blockingreagent (M.O.M. Kit, Vector Laboratories), washed, and sequentiallyequilibrated with MOM protein concentrate/PBS, and MANDYS8 diluted1:1800 in MOM protein concentrate/PBS. Following overnight primaryantibody incubation at 4° C., sections were washed, incubated with MOMbiotinylated anti-mouse IgG, washed, and detection completed withincubation of Vector fluorescein-avidin DCS. Nuclei were counterstainedwith propidium iodide (Molecular Probes) prior to cover slipping withVectashield.

Example 2—Results

Exon 44 Deleted DMD Patient iPSC-Derived Cardiomyocytes ExpressDystrophin after CRISPR Cas9 Mediated Genome Editing by Exon Skipping.

A common hot spot mutation region in DMD patients is the deletion ofexon 44, which leads to skipping of exon 45 in approximately ˜12% of DMDpatients. The DMD patient PBMC-derived iPSCs (TX11) used in this studyhave an exon 44 deletion, which introduces a premature termination codonwithin exon 45 and subsequently disrupts the open reading frame (ORF) ofthe DMD gene. To restore the ORF of the TX11 DMD patient iPSCs, a singleguide RNA was used to disrupt the splicing acceptor of exon 45, whichresults in splicing of exons 43 to 46 and restoration of the proteinreading frame (FIGS. 1A, 3A). The sequence of the intron 44 and exon 45junction contains a 33 base pair conserved region in mouse and humangenomes (FIGS. 1B, 3B). To test sgRNA efficiency within this region,sgRNAs were designed to target the splicing junction sites of exon 43and 45 (FIGS. 1B, 3C, 3D). The cleavage efficiency of these gRNAs wasvalidated in both mouse 10T1/2 cells and human 293 cells. By T7E1 assay,it was demonstrated that 4 sgRNAs (G3, G4, G5 and G6) efficiently causeDNA cleavage at DMD exon 43 or 45 locus (FIGS. 1C, 3E, 3F; Tables 5, 6).

The sgRNA3 (G3) was then tested on TX11 DMD patient iPSCs and observedgenome cleavage at DMD exon 45 locus by T7E1 assay (FIG. 1D). 48 singleclones were picked from a pool of edited TX11 iPSCs mixture andsequenced the edited genomic region. Out of 48 clones, two clones withan abolished splicing acceptor site were observed, which should restorethe DMD ORF (FIG. 1E). Exon 45 skipped TX11 iPSC single clones weredifferentiated using a previously described method (see Materials andMethods section). Restoration of dystrophin protein expression in theclones was confirmed by Western blot analysis (FIG. 1F). To furtherassess the efficiency of the sgRNAs in vivo, exon 45 sgRNA3 (G3) andsgRNA4 (G4) were then cloned into AAV9-TRISPR-CK8 plasmid (FIG. 1G).Efficiency of the two sgRNAs was evaluated in mouse C2C12 cells, thegenome cleavage was observed at DMD exon 45 locus by T7E1 assay (FIG.1H).

Exon 44 Deleted DMD Patient iPSC-Derived Cardiomyocytes ExpressDystrophin after CRISPR/Cas9 Mediated Genome Editing by Exon Skipping.

iPSCs from DMD patients (TX11 and TX12) that have deletion of exon 44were then generated by reprogramming PBMCs derived from the patients(FIGS. 4A, 4B). Exon 45 sgRNA3 (G3) was tested on TX11 DMD patient iPSCsand 48 single clones were picked from a pool of edited TX11 iPSCsmixture. The clones were then differentiated using a previouslydescribed method (see Materials and Methods section). Restoration ofdystrophin protein expression in the clones was confirmed by Westernblot analysis (FIG. 1F). The edited genomic region was sequenced. Out of48 clones, two clones with an abolished splicing acceptor site wereobserved, which should restore the DMD ORF (FIG. 1E). Exon 43 sgRNA4(E43G4), exon 43 sgRNA6 (E43g6) and exon 45 sgRNA4 (E45g4) was thentested on TX11 DMD patient iPSCs. The restoration of dystrophin in theseTX11 DMD patient iPSCs was confirmed by Western blot analysis andimmunostaining (FIGS. 4C, 4D).

Humanized DMD ΔEx44 Mouse Model Recapitulates Muscle DystrophyPhenotype.

To investigate CRISPR/Cas9-mediated exon 45 skipping in vivo, a mimic ofthe human “hot spot” region was generated in a mouse model by deletingthe exon 44 using CRISPR/Cas9 system directed by 2 single guide RNAs(sgRNA) (FIGS. 2A, 5A). sgRNAs targeting 5′ end (In44-1, In44-2 orIn44-3) and 3′ end (In44-4, In44-5, In44-6) of Dmd exon 44 were designedand validated (Tables 5, 6). A T7E1 assay showed different CRISPR/Cas9cleavage efficiencies at Dmd intron 44 in 10T1/2 mouse fibroblasts (FIG.2B). C57BL/6 zygotes were co-injected with in vitro transcribed Cas9mRNA and in vitro transcribed In44-2 and In44-6 sgRNA and re-implantedinto pseudo-pregnant females.

The deletion of Dmd exon 44 was confirmed by DNA genotyping (FIG. 2C).The deletion of exon 44 placed the dystrophin gene out of frame leadingto the absence of dystrophin protein in skeletal muscle and heart (FIGS.2E and 2F). Mice lacking exon 44 showed pronounced dystrophic musclechanges by 1-month of age (FIG. 2G). Mice lacking exon 44 showeddecreased muscle function (FIGS. 5C, 5D, 5E). Serum analysis of ΔEx44mice showed a significant increase of creatine kinase (CK) activity,which is a sign of muscle damage (FIGS. 2D, 5B). Taken together,dystrophin protein expression, muscle histology and serum creatinekinase activity, and muscle function validated dystrophic phenotype ofΔEx44 mouse model.

Correction of DMD Exon 44 Deletion in Mice by Intramuscular AAV9Delivery of Gene Editing Components.

To further assess the efficiency of the sgRNAs in vivo, exon 45 sgRNA3(G3) and sgRNA4 (G4) were then cloned into AAV9-TRISPR-CK8 plasmid (FIG.1G). Efficiency of the two sgRNAs was evaluated in mouse C2C12 cells,the genome cleavage was observed at DMD exon 45 locus by T7E1 assay(FIG. 1H). The AAV9-Cas9 and AAV9-exon 45-sgRNA4 were then injected intothe TA muscle of Δ44 DMD mice. 3 weeks after intramuscular injection,restoration of dystrophin protein expression in the TA muscle of Δ44 DMDmice was confirmed by immunostaining (FIG. 6A), histology (FIG. 6B), andWestern blot analysis (FIG. 6C). The genome cleavage of exon 45 locus inthe corrected Δ44 DMD mice was validated in a T7E1 assay (FIG. 6D).RT-PCR analysis of the TA muscles from WT, Δ44 and Δ44 mice 3 weeksafter intramuscular injection of AAV9-Cas9/exon45-sgRNA4 confirmedskipping of exon 45 (FIG. 6E). RT-PCR sequence analysis of TOPO-TA(topoisomerase-based thymidine to adenosine) generated clones revealedboth exon skipping of exon 45 and reframing of exon 45 contributed tothe restoration of dystrophin open reading frame (FIGS. 6F, 6G).

Systemic AAV9 Delivery of Gene Editing Components to Δ44 Mice RescuesDystrophin Expression.

Rescue of the disease phenotype was then tested by intraperitonealinjection of AAV9-Cas9 and AAV9-exon45-sgRNA4 into Δ44 mice, whichallowed for systemic distribution of the AAV9 vectors. 4 weeks aftersystemic delivery, the restoration of dystrophin protein expression inthe TA, diaphragm, triceps, and cardiac muscles of Δ44 DMD mice wasconfirmed by Western blot analysis (FIG. 7A), histology, andimmunostaining (FIG. 7B). An optimized sgRNA to Cas9 ratio was confirmedby improved correction efficiency in the Δ44 DMD mice. Serum creatinekinase level was decreased in the corrected Δ44 DMD mice (FIG. 7C).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the disclosure. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

VIII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A mouse whose genome comprises a deletion of exon 44 of thedystrophin gene resulting in an out of frame shift and a premature stopcodon in exon
 45. 2. The mouse of claim 1, further comprising a reportergene located downstream of and in frame with exon 79 of the dystrophingene, and upstream of a dystrophin 3′-UTR, wherein the reporter gene isexpressed when exon 79 is translated in frame with exon
 43. 3. The mouseof claim 2, wherein the reporter gene is luciferase. 4.-6. (canceled) 7.The mouse of claim 1, wherein the mouse is heterozygous for thedeletion.
 8. The mouse of claim 1, wherein the mouse is homozygous forthe deletion.
 9. The mouse of claim 1, wherein the mouse exhibitsincreased creatine kinase levels.
 10. The mouse of claim 1, wherein themouse does not exhibit detectable dystrophin protein in heart orskeletal muscle. 11.-20. (canceled)
 21. An isolated cell obtained fromthe mouse of claim
 1. 22.-28. (canceled)
 29. A mouse produced by amethod comprising the steps of: (a) contacting a fertilized oocyte withCRISPR/Cas9 elements and two single guide RNA (sgRNA) targetingsequences flanking exon 44, thereby creating a modified oocyte, whereindeletion of exon 44 by CRISPR/Cas9 results in an out of frame shift anda premature stop codon in exon 45; (b) transferring the modified oocyteinto a recipient female.
 30. A method of screening a candidate substancefor DMD exon-skipping activity comprising: (a) contacting a mouseaccording to claim 1 with a candidate substance; and (b) assessing inframe transcription and/or translation of exon 79, wherein the presenceof in frame transcription and/or translation of exon 79 indicates thecandidate substance exhibits exon-skipping activity.
 31. The method ofclaim 30, wherein the mouse does not exhibit detectable dystrophinprotein in heart or skeletal muscle. 32.-39. (canceled)
 40. An isolatednucleic acid encoding a DMD guide RNA and comprising the sequence as setforth in any one of SEQ ID NO. 1-8 or 27-38. 41-44. (canceled)
 45. Amethod of correcting a dystrophin gene defect in Exon 45 of the DMD genein a subject comprising contacting a cell in the subject with Cpf1 orCas9 and a DMD guide RNA as defined in claim R421140, resulting inselective skipping of a mutant DMD exon.
 46. The method of claim 45,wherein the cell is a muscle cell, a satellite cell, or an iPSC/iCM. 47.The method of claim 45, wherein Cpf1 and/or DMD guide RNA are providedto the cell through expression from one or more expression vectorscoding therefor.
 48. The method of claim 47, wherein the expressionvector is a viral vector.
 49. The method of claim 48, wherein the viralvector is an adeno-associated viral vector.
 50. The method of claim 47,wherein the expression vector is a non-viral vector.
 51. The method ofclaim 45, wherein Cpf1 or Cas9 is provided to the cell as naked plasmidDNA or chemically-modified mRNA. 52-59. (canceled)
 60. The method ofclaim 45, wherein the correction is permanent skipping of the mutant DMDexon. 61.-62. (canceled)